This invention relates to the selective separation of glycosylated hemoglobin (Hb Al) from non-glycosylated hemoglobin in human blood. Another aspect of this invention relates to the selective separation of non-glycosylated hemoglobin from human blood using an ion-exchange system which does not require rigid control of pH and ionic strength and which shows little temperature dependence in the range of from about 15.degree.-37.degree. C. Still another aspect of this invention relates to a method for separating glycosylated hemoglobin from non-glycosylated hemoglobin and quantitatively determining the fractional amount of glycosylated hemoglobin present in human blood through use of a reference material prepared from human blood.
Throughout this circulatory life of the human red cell, glycosylated hemoglobin is formed continuously by the adduction of glucose to the N-terminal of the hemoglobin beta chain. This process, which is non-enzymatic, reflects the average exposure of hemoglobin to glucose over an extended period. Several classical studies have shown that glycosylated hemoglobin in diabetic subjects can be elevated 2-3 fold over the levels found in normal individuals. [Trivelli, L. A., et al., 1971, New Eng. J. Med. 284: 353; Gonen, B., and Rubenstein, A. H., 1978, Diabetologia 15: 1; and Gabbay, K. H., et al., 1977, J. Clin. Endocrinol. Metab. 44: 859]. These cited investigators have recommanded that glycosylated hemoglobin serve as an indicator of diabetic control since the glycosylated hemoglobin levels approach normal values for diabetics responding to treatment. Historically, fastening plasma glucose and urinary glucose tests have been employed as measures of diabetic control. The use of glycosylated hemoglobin determinations offers several advantages over these methods: (1) the glycosylated hemoglobin level is unaffected by the ingestion of a recent meal; (2) it appears quite stable in the blood; and (3) it reflects the average blood glucose level over an extended period (3-4 weeks) rather than at a single time point, thus providing a better criterion of diabetic control. Thus, an accurate, reproducible and dependable in vitro quantitative test for glycosylated hemoglobin is important in medicine for the clinical management of diabetic patients.
Glycosylated hemoglobin has been defined operationally as the fast fraction hemoglobins (Hb, Ala, Alb, Alc) which elute first during column chromatography with cation-exchange resin. The non-glycosylated hemoglobin, which consists of the bulk of the hemoglobin, remains attached to the resin and can be removed by lowering the pH or raising the ionic strength of the eluting buffer. In the past, a carboxy derivative of cellulose or polystryene has been commonly employed as the ion-exchange resin. Elution of the glycosylated hemoglobin was accomplished by use of a phosphate buffer containing cyanide. These methods are disclosed in Trivelli, L. A., et al., 1971, New Eng. J. Med. 284: 353; and Gabbay, K. H., et al., 1977, J. Clin. Endocrinol. Metab. 44: 859. Other methods used to separate glycosylated hemoglobin include high-performance cation-exchange chromatography and electrophoresis. These latter procedures require expensive equipment and usually prove too slow and cumbersome for practical uses.
Difficulties can occur in practicing these methods for separating glycosylated hemoglobin from non-glycosylated hemoglobin. For proper quantitative determinations, the composition, pH and ionic strength of the eluting buffer must be maintained within narrow limits. More importantly, temperature control is critical, usually being confined to a range of 21.degree.-24.degree. C. Such rigid limitations on a procedure can cause operating difficulties beyond the capabilities of many clinical laboratories. Furthermore, substantial amounts of cyanide are used in the eluting buffer and represent a hazard to the user. These problems are described by Simon, M., and Eissler, J., 1980, Diabetes 29: 467; Rand, P. G., and Nelson, C., 1980, Clin. Chem. 26: 1209; and Schellekens, A. P. M., et al., 1981, Clin. Chem. 27: 94.
Thus, a need exists for an ion-exchange system and method for determining glycosylated hemoglobin in human blood which offers ease of handling, has little temperature dependence, does not require rigid control of pH and ionic strength, and uses a minimal amount of cyanide.